Structural Origin of the Large Redox-Linked Reorganization in the 2-Oxoglutarate Dependent Oxygenase, TauD

Abstract

2-Oxoglutarate (2OG)-dependent oxygenases catalyze a wide range of chemical transformations via C−H bond activation. Prior studies raised the question of whether substrate hydroxylation by these enzymes occurs via a hydroxyl rebound or alkoxide mechanism and highlighted the need to understand the thermodynamic properties of transient intermediates.A recent spectroelectrochemical investigation of the 2OG-dependent oxygenase, taurine hydroxylase (TauD), revealed a strong link between the redox potential of the Fe(II)/Fe(III) couple and conformational changes of the enzyme. In this study, we show that the redox potential of wild-type TauD varies by 468 mV between the reduction of 2OG-Fe(III)-TauD (−272 mV) and oxidation of 2OG-Fe(II)-TauD (+196 mV). We use active site variants to investigate the structural origin of the redox-linked reorganization and the contributions of the metal-bound residues to the dynamic tuning of the redox potential of TauD. Time-dependent redox titrations show that reorganization occurs as a multistep process. Transient optical absorption and infrared spectroelectrochemistry show that substitution of any metal ligand alters the kinetics and thermodynamics of the reorganization. The H99A variant shows the largest net redox change relative to the wild-type protein, suggesting that redox-coupled protonation of H99 is required for high redox potentials of the metal. The D101Q and H255Q variants also suppress the conformational change, supporting their involvement in the structural rearrangement. Similar redox-linked conformational changes are observed in another 2OG dependent oxygenase, ethylene-forming enzyme, indicating that dynamic structural flexibility and the associated thermodynamic tuning may be a common phenomenon in this family of enzymes.

Glutarate Hydroxylation by the Carbon Starvation-Induced Protein D: A Computational Study into the Stereo- and Regioselectivities of the Reaction

“…The three reactant complexes Re 1 , Re 2 , and Re 3 have a short Fe–O distance of 1.65 Å, which is expected for the double bond configuration of this complex. These values compare well with the experimental work on analogous nonheme iron enzymes − and are similar to related nonheme iron enzyme calculations and biomimetic model complexes calculated previously. − Moreover, experimental Mössbauer and resonance Raman spectroscopy on analogous nonheme iron dioxygenases found similar bond lengths and typically a quintet spin ground state for the iron­(IV)-oxo species. − In all three reactant structures, the axial histidine ligand was positioned at around 2.06–2.09 Å from the iron (Fe–N ax distance) in the quintet spin state structures.…”

“…Because of the difficulties encountered in trapping and characterizing short-lived catalytic cycle intermediates in CsiD and the lack of information on the details of its enzymatic reaction mechanism, we decided to perform a computational study. Previous experimental studies on related enzymes showed that α-ketoglutarate-dependent dioxygenases often operate through the formation of a high-valent iron­(IV)-oxo species as the active oxidant. − Indeed, computational studies on nonheme iron/α-KG-dependent dioxygenases the initial reaction of iron­(II) with dioxygen in the presence of a bound α-KG molecule gives an iron­(IV)-oxo species, succinate and releases a CO 2 molecule with large exothermicity. − The iron­(IV)-oxo species of several nonheme iron dioxygenases has been characterized by UV–vis absorption, resonance Raman, electron paramagnetic resonance, and Mössbauer spectroscopy methods. − , All of these studies identify the iron­(IV)-oxo species as the active oxidant in the substrate activation reaction. Our study therefore starts from the iron­(IV)-oxo species of CsiD, where we took the 6HL9 pdb file and inserted a glutarate ion into the substrate binding pocket with the Autodock Vina program .…”

“…71−76 The iron(IV)-oxo species of several nonheme iron dioxygenases has been characterized by UV− vis absorption, resonance Raman, electron paramagnetic resonance, and Mossbauer spectroscopy methods. [8][9][10][11][12][13][14]37 All of these studies identify the iron(IV)-oxo species as the active oxidant in the substrate activation reaction. Our study therefore starts from the iron(IV)-oxo species of CsiD, where we took the 6HL9 pdb file and inserted a glutarate ion into the substrate binding pocket with the Autodock Vina program.…”

“…These enzymes use α-ketoglutarate (α-KG) as a cosubstrate that in the initial reaction step with dioxygen on the iron cofactor is converted into succinate and CO 2 and generates a high-valent iron­(IV)-oxo species. The latter, for instance, was characterized for the nonheme iron dioxygenase taurine/α-ketoglutarate dioxygenase with a range of spectroscopic techniques. − It has been proposed to be the active oxidant for many nonheme iron enzymes, and generally, the iron­(IV)-oxo reacts through oxygen atom transfer to substrates, thereby converting an aliphatic group into an alcohol. In addition, this enzyme family also has been shown to perform substrate halogenation, ring formation, desaturation, and epoxidation reactions, − although substrate hydroxylation is the most abundant reaction type conducted by the iron­(II)/α-KG-dependent oxygenases.…”

N-Nitrosation Mechanism Catalyzed by Non-heme Iron-Containing Enzyme SznF Involving Intramolecular Oxidative Rearrangement

“…Based on the electron-transfer processes in the catalytic reactions, these non-heme iron-containing enzymes can be roughly classified into two main categories, one performs two-electron oxidation of the substrates and the other fully couples the reduction of dioxygen to the four-electron oxidation of the substrates. Fe II /2OG-dependent oxygenases fit into the first category, which carry out a variety of two-electron oxidation processes on the substrates, leading to hydroxylation, − halogenation, , dehydrogenation, ,− and cyclization products. − In Fe II /2OG-dependent oxygenases, the substrates do not coordinate to the iron center directly; therefore, the oxidation reaction has to be accomplished through a highly active Fe IV O core. They consequently require cosubstrate 2OG as an exogenous electron resource to provide two extra electrons to reduce dioxygen and to assist to generate the highly active Fe IV O core.…”